Composite devices incorporating biological material and methods

ABSTRACT

The present invention provides composite biological devices that include biological material as an integral component thereof. The devices can be used for measuring metals, for example, particularly toxic metals such as mercury.

This application is a U.S. National Stage Application which claimspriority to International Application No. PCT/US99/21581, filed Sep. 17,1999, and also claims priority to U.S. Provisional Application No.60/100,914, filed Sep. 17, 1998, which are incorporated herein byreference.

STATEMENT OF GOVERNMENT RIGHTS

The present invention was made with support of the National ScienceFoundation under Grant No. 9424063. The government may have certainrights in this invention.

FIELD OF THE INVENTION

The present inventions provides composite devices that includebiological material as an integral element, as well as methods of theiruse and preparation. Such devices can be used for screening drugs,chemical catalysis, implanting (into mammals, birds, fish),bioelectronic applications, biosensor applications (including single useindictors and sensors that can be released into the environment). Anexample of this concept is a device for measuring environmentalcontaminants such as metals.

BACKGROUND OF THE INVENTION

Metabolically active biological materials (e.g., cells) are phenomenalbiochemical catalysts capable of carrying out sequential, stereospecificbiochemical reactions, and can function as sensitive biosensors. Thereare significant potential industrial, biomedical, and environmental usesof metabolically active biological materials. They can be used for avariety of purposes such as detecting and/or measuring the amount of anenvironmental contaminant, particularly metals.

Metal contamination, particularly mercury, arsenic, cadmium, chromium,and nickel contamination, continues to be a public health andenvironmental problem. Conventional chemical detection techniquesinclude atomic absorption spectrophotometery, ion chromatography, gaschromatography, mass spectrometry, as well as cold-vapor atomicabsorption or cold-vapor atomic fluorescence spectroscopy. At least someof these techniques can be highly sensitive but complex to perform andexpensive in terms of equipment and training. Furthermore, thesetechniques must typically be conducted in the laboratory. Plus, thesetechniques do not always reflect the true biological availability oftoxic metals in a system.

Microorganisms that quantitatively detect toxins in the environmentoffer a less expensive alternative to conventional methods. For example,microbial biosensors aimed at measuring the bioavailability of mercuryhave been developed as an alternative to chemical or physical analysis.U.S. Pat. No. 5,612,184 (Rosson) discloses a device for the detection ofmercury in water using an aqueous suspension of recombinant biosensorymicroorganism cells containing a lux bioluminescence gene. The cells arebioluminescent in the presence of Hg²⁺ ions and/or monomethyl mercury.The resultant bioluminescence can be detected using a variety of means,e.g., photographic film, photomultiplier, photodiode, or scintillationcounter. However, this patent only discloses the use of such a suspendedcell biosensor for the detection of mercury in water. Furthermore,suspended cell biosensors are limited because of handling difficultiesand short useful life of the cell stock solution.

Immobilization of cells for the biodetection of contaminants in aqueousenvironments offers advantages over the use of suspended cell systems.Immobilized cells are easy to handle, can remain viable for long periodsof time, and show excellent plasmid retention. However, immobilizationmethods for use in biosensors have focused on reusable detection methodswhere the immobilized cells are used repeatedly. In such methods,control over immobilized cell stability and cell outgrowth becomeconsiderable problems together with slow biosensor response times.

SUMMARY OF THE INVENTION

The present invention is directed to composite devices that incorporateone or more biological materials (e.g., microorganism such asprokaryotic, eukaryotic, or archean organisms, as well as mammaliancells, blood cells, avian cells, plant cells, insect cells, spores,phages, viruses, etc.) as an integral element. The biological materialis preferably viable, i.e., metabolically active, and substantiallypermanently immobilized within the devices. The present inventionsignificantly expands on the potential industrial, biomedical, andenvironmental uses of metabolically active biological materials (such ascells) by incorporating them as an integral component of compositedevices. For example, these devices can be used for a variety ofpurposes such as detecting and/or measuring the amount of anenvironmental contaminant, drug, organic or inorganic compound thatindicates the quality or purity of air, water, soil, or foods.

In one embodiment, the present invention provides a composite biologicaldevice. The device includes a biostructure that includes at least onemetabolically active biological material as an integral componentthereof. At least a portion of the biostructure includes a nonporouslatex-derived material. Preferably, the biostructure includes at leastone layer of a porous latex-derived material and at least one layer of anonporous latex-derived material. The nonporous material can be used tocreate a variety of structures within the device. For example, nonporousmaterial can define at least one channel or at least one well.

The biostructures of the devices of the present invention may be selfsupporting or may be disposed on a substrate. Preferably, thebiostructures are very thin. For example, they are preferably, nogreater than about 500 microns in thickness, more preferably, no greaterthan about 100 microns in thickness, and most preferably, no greaterthan about 10 microns. For certain embodiments, the entire device is nogreater than about 500 microns in thickness.

In another embodiment, the present invention provides a compositebiological device that includes a 3-dimensional porous latex-derivedbiostructure having at least one metabolically active biologicalmaterial incorporated therein; wherein the biostructure is disposed on aporous substrate.

In yet another embodiment, the present invention provides a compositebiological device that includes a 3-dimensional porous latex-derivedbiostructure having at least one metabolically active biologicalmaterial incorporated therein; wherein the porous latex-derivedbiostructure contains at least two portions of different pore size.

The present invention also provides a method of making a compositebiological device. The method includes depositing at least one latex ina first layer; depositing at least one latex in a second layer on thefirst layer to form a microstructure; depositing at least onemetabolically active biological material separately or in a combinationwith at least one latex such that the biological material isincorporated into the microstructure; wherein at least one of thelatices forms a nonporous component of the microstructure. The methodpreferably involves ink-jet printing with an ink-jet printer.

In a preferred embodiment, the present invention provides a compositebiological device for determining the presence of a metal in a sample.The composite biological device includes a biostructure having at leastone biological material which, upon contact with the metal, produces aresponse and emits a signal. Preferably, the biological materialincludes bacterial cells immobilized (preferably, permanently entrapped)in one or more layers of a polymeric material. Preferably, the cells aregenetically engineered to produce a response, such as luminescence, tothe metal of interest. In certain embodiments, the biostructure isdisposed on a substrate that is capable of detecting the signal. In suchembodiments, the substrate is a photosensitive film or a light-sensitiveelectronic chip, for example.

The present invention also provides a method of determining the presenceof an analyte (e.g., metal) in a sample (e.g., liquid, gas, solid, orsemi-solid sample). The method comprises contacting the sample with adevice as described herein, wherein, upon contact with the analyte, thebiological material produces a response and emits a signal; anddetecting the signal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a cross-sectional schematic of one embodiment of a deviceaccording to the present invention using a photosensitive film as thesupporting substrate.

FIG. 1B is a bottom view of the device of FIG. 1A showing thephotosensitive film.

FIG. 2A is a cross-sectional schematic of an alternative embodiment of adevice according to the present invention using a light-sensitiveelectronic chip as the supporting substrate.

FIG. 2B is a bottom view of the device of FIG. 2A showing thelight-sensitive electronic chip.

FIG. 3 is a cross-sectional schematic of an alternative embodiment of adevice according to the present invention using a solid light-proof orlight impenetrable backing as the supporting substrate.

FIG. 4 is a cross-sectional schematic of an alternative embodiment of adevice according to the present invention that is used to penetrate asolid sample such as fish tissue.

FIG. 5 is a cross-sectional schematic of an alternative embodiment of adevice according to the present invention that includes a cavity forplacement of a solid sample, such as fish tissue.

FIG. 6 is a cross-sectional schematic of an alternative embodiment of adevice according to the present invention that includes a cavity forplacement of a solid sample containing a fluid, such as an absorbent padcontaining fluids from fish tissue.

FIG. 7A is a schematic of an alternative embodiment of a deviceaccording to the present invention that can be used to penetrate throughfish scales, skin, and tissue and remove a core sample of tissue.

FIG. 7B shows the device of FIG. 7A penetrating fish scales, skin, andtissue.

FIG. 8A is a schematic of an alternative embodiment of a deviceaccording to the present invention that can be used to penetrate throughfish scales, skin, and tissue.

FIG. 8B shows the device of FIG. 7A penetrating fish scales, skin, andtissue.

FIG. 9A is a cross-sectional schematic of an alternative embodiment of adevice according to the present invention that incorporates aphotosensitive film as the supporting substrate and isolates the persontaking the sample from the sampled material, thereby preventing thetransfer of disease.

FIG. 9B is a side view of the device of FIG. 9A showing thephotosensitive film.

FIG. 10A is a cross-sectional schematic of an alternative embodiment ofa device according to the present invention that uses a coated fiber inwhich the coating contains immobilized cells.

FIG. 10B shows an incubation pouch containing a photosensitive film usedfor detecting a signal emitted by the cells of the coated fiber of FIG.10A.

FIG. 11A is a cross-section of a monofilament coated with immobilizedcells.

FIG. 11B is a cross-section of a multiple filament thread coated withimmobilized cells.

FIG. 12 is a schematic of an alternative embodiment of a deviceaccording to the present invention that includes a pop-up indicator anda protein or lipid-based glue and immobilized cells containing aprotease reporter gene.

FIG. 13 is a schematic of preferred template assemblies beforecell-coating, after cell coating, and after top-coating. A: Top-view andside-view of template assembly; B: cell-coat patches with surroundingspacer; C: finished top-coated patches.

FIG. 14 is a schematic of a latex biosensor patch containing a viable E.coli coating, a porous sealant coating, and a nonporous wall coating.

FIG. 15 is a schematic of a latex biosensor patch containing a viable E.coli coating, a porous sealant coating, a mercury adsorbent (such asAPDC) layer coating, and a nonporous wall coating.

FIG. 16 is a schematic of a latex biosensor patch containing a viable E.coli coating, a porous sealant coating, a porous channel coating, and anonporous wall and sealant coating.

FIG. 17 is a schematic of a layered porous array of microwells withbidirectional channels.

FIG. 18 provides a chart of luciferase activity of latexfilm-immobilized E. coli HB101 containing mer-lux constructs afterinduction by HgCl₂ in pyruvate buffer. (A) pRB28. (B) pOS14, (C) pOS15.Symbols: (□) 10,000 nM HgCl₂, (∘) 1,000 nM HgCl₂, (Δ) 100 nM HgCl₂, (∇)0 nM HgCl₂, (∘) 1 nM HgCl₂, (+) 0.1 nM HgCl₂, and (x) 0 nM HgCl₂.

FIG. 19 provides a chart of the effect of storage on latex-immobilizedE. coli HB101 (pRB28). Maximum luciferase activity was plotted as afunction of mercury concentration. (A) at −20° C. for 3 month. Symbols:(▪) immobilized cells freshly made, (▴) immobilized cells stored at −20°C. for 3 months in glycerol: PBS buffer (50:50 w/w). (B) at ambienttemperature dry 14 days. Symbols: (▪) immobilized cells freshly made,(▾) immobilized cells stored ambient temperature for 14 days dry.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides devices that include as an integralcomponent thereof biological material, preferably, metabolically activebiological material. By incorporating metabolically active biologicalmaterial (e.g., cells) as an integral component, the compositebiological devices of the present invention expand on the potentialindustrial, biomedical, and environmental uses of such metabolicallyactive biological materials.

The general applications of these composite biological devices are: highthroughput automated drug screening/drug discovery; as compositebiochemical catalysts for the manufacture of drugs, fine chemicals, andfood ingredients; as implantable “tissue-like” structures in mammals,birds, or fish; as composite environmental sensors to detect thepresence of environmental toxins; as bioelectronic devices to store andtransmit information; as inexpensive analytical sensors for clinical,occupational, or environmental monitoring; and as efficient biocatalystsfor municipal and industrial waste treatment.

A particularly useful application of this invention is for screening.This includes automated analysis of the integral biological materials(e.g., cells as the “targets”) for sensitivity (killing, for example, byantibiotics, antiviral compounds, antitumor agents), bioactivity,pathogenicity, virulence, and resistance to chemical and physicalchallenges. These composite biological devices can also be used assensitive indicators of gene expression from the integral biologicalmaterial(s) in response to chemical or physical challenges and in thepresence of other biological materials (such as cell—cell or cell-tissueinteractions). These novel devices are also useful as indicators of themetabolic response of the integral biological material (e.g., measuringthe waste products of cellular metabolism), growth, viability (e.g., thecapability for DNA synthesis), and the ability to be stimulated/respondas the biological materials would in vivo.

Preferably, the devices are inexpensive and disposable. They can be madeof both porous and nonporous materials, preferably latex polymers. Theyinclude a biostructure, which can include one or more layers ofpolymeric material. These layers can be continuous or discontinuous andpattern coated, thereby forming patches or reactive zones. At least aportion of the biostructure includes biological material (i.e.,biomaterial). Preferably, at least a portion of the biostructureincludes a nonporous latex-derived material. The biostructure canoptionally be disposed on a substrate, which for certain embodiments isporous. The biostructure is preferably very thin. Preferably, it is nogreater than about 500 microns thick, more preferably, no greater thanabout 100 microns thick, and most preferably no greater than about 10microns thick. More preferably, the entire device, biostructure andoptional substrate on which the biostructure is disposed is no greaterthan about 500 microns thick.

A preferred embodiment of these composite materials is for thedetermination of the presence of a metal in a sample. Advantageously,the devices can also quantitatively measure the amount of a metal in asample.

Metals that can be detected, typically individually detected, andpreferably quantitatively measured, include inorganic or organic formsof a variety of metals that can be toxic to humans and other species.These include, for example, mercury (typically, in the form of Hg²⁺ ormonomethyl mercury), arsenic (typically in the form of arsenate AsO₄ ³⁻or arsenite AsO₂ ⁻), cadmium (typically in the form of Cd²⁺), antimony(typically in the form of antimonite SbO₂ ⁻), bismuth (typically in theform of Bi³⁺), and copper (typically in the form of Cu²⁺).

Other components (e.g., analytes in a sample of interest) that can bedetected and/or quantitatively measured using the devices of the presentinvention include organic compounds that can be toxic to human, avian,plant, fish, insect, or other species. These include, for example,insecticides, herbicides, polycyclics, nerve gas agents, mutagens,carcinogens, antibiotics, products of combustion (e.g., tobacco smoke,coal combustion, liquid fuel combustion). Such compounds includehydrocarbons (e.g., xylene, toluene, naphthalene), halogenatedhydrocarbons (e.g., trichloroethylene, carbon tetrachloride,chloroform), formaldehyde, ketones, hydrazines, and the like.

The devices can be used to determine the constituents of liquid samplessuch as oil, water, as well as biological fluids such as blood, urine,saliva, tears, extracts of biological tissue, for example. The liquidsamples can be held by an absorbent pad, such as one made of celluloseor sponge. Alternatively and significantly, the devices can be used onsolid (or semi-solid) samples such as biological tissue (e.g., fromseafood, particularly fish, clams, crabs, oysters, for example) as wellas sludge and soil. The devices can also be used on gaseous samples suchas air.

Preferably, and significantly, the devices of the present invention arestable such that the biological material (e.g., cells or microorganisms)remain viable. (i.e., metabolically active). By stable, it is meant thatthey are responsive after at least about 8 hours under ambientconditions when the biostructure is in a hydrated condition (duringand/or prior to use, the cells are hydrated). More preferably, thedevices are stable for at least about 6 months under ambient conditions,and most preferably, indefinitely at a temperature of less than about−10° C., when the biostructure is in a prehydrated (or nonhydrated)condition. For example, devices of the present invention have been shownto remain stable (e.g., maintained 90% of its initial metabolic activitywhen rehydrated) for at least about 4 months at −20° C. in a prehydratedcondition. The devices of the present invention are also preferablyrobust such that they can be handled and transported with little or nodamage. The devices can be flexible. Preferably, they include anindicator coating that does not delaminate from a supporting substrate,craze, or crack.

The devices of the present invention preferably include immobilizedbiological material that form an integral part of the composite device.Typically, such biological material is immobilized in a polymeric layer,which can be in the form of a coating, and is preferably supported on asubstrate (typically, an inert substrate that does not interact orinterfere with the function of the device), although a support substrateis not required. This coating with at least one biological materialtherein is referred to as a “biological material-containing layer” or“biomaterial-containing layer.” As used herein, a layer can becontinuous or discontinuous. A variety of such layers can be combined toform a variety of structures within the device, such as channels, wells,etc.

Preferably, the devices include a multi-layered construction, which isreferred to herein as a 3-dimensional microstructure. A polymeric layerthat includes the immobilized biological material is typically supportedon a substrate (preferably, an inert substrate). This layer forms amicroporous matrix that entraps (typically, permanently entraps) wholeliving cells (and even microorganisms) as an integral element of thedevice, typically without adhering to them. The biological material canbe present in the device in multiple layers if desired. Optionally, andpreferably, the construction includes at least one interlayer oroverlayer (i.e., sealing or sealant layer) of a polymer that does notinclude biological material. An overlayer can help prevent thebiological material from leaving the first polymeric layer uponrehydration of the biological material with water or a water-basedsolution. Other layers, which may form channels, wells, or otherstructures in an array, are possible as well. Typically, thesestructures are formed by nonporous material, preferably, nonporouslatex-derived polymers. Thus, as used herein, a composite device caninclude one or more biological material-containing polymeric layers andone or more polymeric layers that do not include biological materialthat can be interspersed between the biological material-containinglayers or as overlayers.

In certain embodiments, the biostructures preferably include no greaterthan about 75% by volume of biological material, and more preferably, nogreater than about 50% by volume of biological material. Cellsimmobilized in biostructures according to the present inventiontypically maintain at least about 80% of the original culturability, andpreferably have rehydrated culturability that is similar or higher thanthat of suspended cells when compared over 15 days.

The polymeric layers of the device can be porous or nonporous.Preferably, if they contain biological material, they are porous, and ifthey do not, they are less porous, and even nonporous. For certainembodiments, the biostructure includes at least two different porouspolymers of different pore sizes.

The porosity of a latex polymeric layer results from the fluid-filledspaces that remain between the polymer particles after polymer particlecoalescence. Low porosity or nonporous latex layers are typically formedby latices with very rapid and complete polymer particle coalescence.Examples of these latices include latex paints. Such nonporous polymerscan be pattern coated to form nonporous channels, reservoirs, and wells,for example.

The porosity of latex polymer layers can be controlled by a variety ofmethods that arrest polymer particle coalescence. Some degree of polymercoalescence or “welding” is typically required for film formation and toimmobilize the biological material. Various methods exist to arrest orcontrol the degree of polymer particle coalescence to obtain optimalporosity. For example, the degree of polymer particle coalescence can bealtered by the presence of carbohydrates, or surface active agents, orby polymer particle composition, film formation temperature, and/ordrying conditions.

Latex porosity is commonly measured by monitoring the rate of diffusionof a nonbinding, easily detected, low molecular mass molecule through alatex film using a diffusion apparatus. These indicator moleculesrapidly diffuse through (from one side to the other) a highly porouslatex film. They diffuse slowly through low porosity films.

Each layer, which may be continuous or discontinuous, may contain one ormore polymer. Each polymer used is preferably derived from a latex(e.g., water delivered polymer particles), whether it be naturallyoccurring or synthetic. Other non-latex-derived polymers can also beused if desired. The polymer particles may be monodispersed (all ofsimilar size), polydispersed (broad polymer particle distribution), orspecific combinations thereof. The polymers can include, for example,acrylate polymers, vinyl acetate polymers, styrene polymers, butadienepolymers, carboxylate polymers, and blends or copolymers thereof. Asused herein a copolymer is a polymer of two or more different types ofpolymers (including copolymers, terpolymers, tetrapolymers, etc.). Thepolymers may or may not be cross-linked. Suitable polymers arecommercially available from Rohm and Haas of Philadelphia, Pa., Dupontof Wilmington, Del., H.B. Fuller Co. of Minneapolis, Minn., and GenCorp.of Magadore, Ohio, for example. Preferably, the polymeric material usedfor immobilizing (i.e., entrapping) biological material includes anacrylic/vinyl acetate copolymer. Preferably, the polymeric material usedas an intervening or overlayer includes an acrylic/vinyl acetatecopolymer.

The polymeric layers (both biological material-containing layer(s) andintervening or overlayer(s) that do not include biological material) canalso include additives for various purposes, such as absorbingundesirable material, preventing microbial contamination, and increasingsensitivity. Such additives include, for example, a salt, a pigment, anadsorbent, a liquid crystal, a porosity modifier, a chelating agent, anutrient, a surfactant, a dye, a photoreactive compound, an antibiotic,an antimicrobial, a bacteriostatic compound, an enzyme, anosmoprotectant, a biopolymer, a metals, a chemical catalyst, or acombination thereof.

Examples of such additives include, but are not limited to: salts suchas NaCl, NiCl, K₂HPO₄, KH₂PO₄, calcium, magnesium, sodium and potassiumcarbonates; porosity modifiers such as glycerol, glucose, and sucrose;adsorbents such as CaCO₃, CaSO₄, MgSO₄; nutrients such as amino acids(e.g., cysteine) and carbohydrates; pigments such as TiO₂; dyes such asX-gal (5-bromo-4-chloro-3 indolyl-β-D-galactoside), blue dextran, andResazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide; chelating agents suchas EDTA and ammonium pyrrolidine dithiocarbamate (APDC); surfactantssuch as FLUORAD FC 430 (3M Co., St. Paul, Minn.); liquid crystals suchas p-methoxy benzyliden-p′-n-butoxyaniline (MBBA); enzymes such asperoxidase; photoreactive compounds such as silver halides;bacteriostatic compounds such as NaF; antibiotics such as kanamycin orampicillin; antimicrobial agents such 1,2-benzisothiazolin-3-one (ICIbiocides, Wilmington, Del.); osmoprotectants such as sucrose ortrehalose or glycerol; biopolymers such as gelatin; metals; and chemicalcatalysts. Preferably, the biomaterial-containing layer(s) includeglycerol. Preferably, the other polymeric layer(s) include glycerol,bacteriostatic compounds, antibiotics, antimicrobial agents, and/orcarbohydrates.

The polymeric layers (both biological material-containing layer(s) andintervening or overlayer(s) that do not include biological material) canalso include physical components that can function as detectors ortransmitters. These can include wires, optical fibers, electroniccomponents such as chips, etc. These may or may not be in direct contactwith the biological material, although preferably, a transmitter isadjacent to or in direct physical contact with the biological material.

Preferably, the biological material includes one or more species ofprokaryotic, eukaryotic, or archean organisms as homogeneous cellpopulations, mixtures of microorganisms, consortia, mixed-cultures, orunspeciated naturally occurring microbial populations with a definedcharacteristic. The biological material can include mammalian cells,blood cells, bacterial cells, avian cells, plant cells, insect cells,spores (e.g., Bacillus subtilis), phages (e.g., lambda bacteriophage),viruses (e.g., HIV, HTLV), etc. The biological material can be in theform of cell clumps or cell mats (i.e., a number of different cellsliving together in some sort of structure), for example. Examples ofsuitable cells include bacterial cells such as E. coli, Bacillus, andStreptomyces, Thermotoga, archean cells such as Pyrococcus, eukaryoticcells such as yeast and Penicillium, as well as plant cells. For certainpreferred embodiments, the biological material includes bacterial,yeast, or fungal cells, which may optionally be recombinant.

The biological material is preferably genetically engineered to producea response, such as a mechanical or chemical response, and thereby emita signal that can be detected. They are also preferably optimized fordesiccation tolerance. Examples of suitable responses include, but arenot limited to; emission of light (e.g., luminescence or fluorescence);production of an enzyme, metabolite, or other detectable chemical;evolution of heat; a change in H⁺ or OH⁻ concentration; a change inthermal or electrical conductivity; a change in pressure; production ofoxygen, a change in a reactive radical concentration; or a change intensile or compressive stress. Such response is typically produced uponcontact with an analyte in a sample being analyzed. For example, aresponse occurs upon contact with a metal being detected. Significantly,many of these responses can be measured such that the material beingdetected can be quantitatively measured. Such responses can betransmitted to a detector, optionally with the aid of a transmitter. Thedetector and optional transmitter may be part of the device, eitherforming a part of the biostructure or incorporated into a substrate onwhich the biostructure is disposed.

Preferably, the biological material is recombinant E. coli, Bacillus, orStreptomyces cells that include a metal resistant promoter, such as amercury resistant promoter, and a reporter gene encoding a protein suchas, for example, luciferase, protease, β-galactosidase, alkalinephosphotase, or green fluorescent protein. Preferably, the biologicalmaterial includes a bioluminescent operator/promoter mer-lux plasmid,although other operator/promoter constructs can be used includingars-lux, smt-lux, and cad-lux.

Several mer-lux plasmid constructs are known. The constructs made bySelifonova et al. (Appl. Environ. Microbiol., 59, 3083–3090, (1993)) areparticularly useful in that each construct has been tested extensivelyfor mercury sensitivity in suspended cultures under differentconditions. These plasmids are pRB28, pOS14, and pOS15. They all codefor luciferase activity (luxCDBE) but differ in the subset of mer genesfused to the lux gene. pRB28 contains merR (the mer repressor gene) anda truncated merT (one of the mer transporter genes). A second construct,pOS14, contains merR and the complete set of mercury transport genesmerT, merP, and merC. The third construct, pOS15, contains merRTPC, thereductase gene (merA) and a second regulatory gene (merD). Induction ofthe mer operon by inorganic mercury results in the production ofluciferase which can be assayed by the ATP-dependent emission ofphotons.

Preferably, the biostructure (preferably, in the form of a 3-dimensionalmicrostructure or array) is supported on a substrate. The substrate caninterface with the biostructure if it includes a detector ortransmitter. Typically, however, the substrate is an inert substrate,which is one that does not take part in or interfere in the function ofthe device. The substrate can be in a wide variety of forms, such as afilm, wire, membrane, filament, foam, etc., including combinations ofsuch materials. It can be transparent or translucent. It can be made ofa wide variety of materials, which may be porous or nonporous, syntheticor naturally occurring, including metals, glasses, ceramics, and organicpolymers (e.g., nylon, polyester, polycarbonate, and polyacetate).Examples of substrates include paper, woven or nonwoven fiber mats,plastic sheets, etc. The substrate can include electronic components,such as electrodes, semiconductor devices.

In particularly preferred embodiments, the substrate enables detectionand/or measurement of the metal by monitoring the signal produced uponthe biological material responding to contact with the metal (e.g.,luminescence). For such preferred embodiments, the supporting substratecan be a photosensitive film or a light-sensitive electronic chip, forexample. Alternatively, the substrate can merely support or protect thebiological material and not take part in the detection of the metal. Forsuch embodiments, the supporting substrate can be a solid, lightimpenetrable backing, for example. In such embodiments in which thesupporting substrate does not detect the signal emitted by thebiological material, the biological material would need to be broughtinto close proximity to a detector. Such a detector could be, forexample, a photosensitive film that does not have a cell-containingindicator coating thereon, a scintillation counter, or light meter. Thisoccurs in the embodiments described below with respect to FIGS. 3, 8,and 10.

The methods of detection include various detection mechanisms. Forexample, such methods can involve detecting light fluorescence as fromexpression of Lux, a fluorescent protein, detecting a hydrolytic enzymeactivity such as protease activity, detecting the production of ametabolite, detecting the evolution of heat, detecting a change in H⁺,OH⁻, or reactive radical concentration change, detecting the evolutionof a gas such as carbon dioxide, detecting the utilization or depletionof a substrate such as glucose, and/or detecting a change of color. Apreferred method is the expression of Lux.

The polymeric layers, with or without biological material incorporatedtherein, may be formed, for example, by a wide variety of methods,including, for example, draw down coating, slot coating, die coating,spin coating, gravure coating, or piezo-electric or acoustic printing(e.g., inkjet or laser jet printing having piezo-electric or acousticpumps). Typically, the biological material-containing layer(s) are driedprior to the overlayer(s) being applied. A typical coating process of apolymeric layer on a substrate can be carried out at temperaturesvarying from about 4° C. to about 95° C. The coating method preferablyprovides good control over biological material distribution, and coatingthickness which leads to easily standardized responses or measurements.Alternatively, however, the layers can be simultaneously coated orcoated sequentially without intervening drying steps, if so desired. Thelayers may also be pattern coated.

Pattern coating of a rapidly coalescing latex facilitates formation ofdevice microstructures consisting of, for example, nonporous latexwalls, dams, or barriers to restrict gas and liquid flow or diffusion.This pattern coating method can be repeated multiple times to depositnonporous latex polymer to predetermined thickness or height. Using thismethod, device structures such as channels, reservoirs, microwells,etc., can be made. This same method can also be used to generate complexthree-dimensional (3-D) interconnected arrays of channels linkingreactive zones, which are regions of the pattern coating containingintegral biological material (preferably, substantially permanentlyentrapped biomaterial). One example is shown in FIG. 17.

A particularly preferred method of forming the biostructures of thedevices of the present invention is through the use of piezo-electric oracoustic printing (e.g., ink jet or laser jet printing). This method,compared to rod, bar, or slot coating methods can immobilize biomaterialin high resolution multilayer microstructures, which can be in the formof a patch, of such high density (e.g., number per unit area) and highspecific activity (e.g., number of cells per microstructure) thatremarkable gains can be realized in biosensor sensitivity, biocatalystvolumetric activity, screening sensitivity, and productivity. Thistechnique can eject or jet biological material in pico-liter sizeddroplets. Polymeric material can be combined with the biologicalmaterial or applied separately by using two fusing streams. Millions ofsingle droplets can be deposited in ultra-high densities (e.g., greaterthan about 1000 dots per inch (dpi)) at very high rates.

A suitable printing apparatus may include ink-jet heads containingseveral rows of nozzles (e.g., 32 nozzles in 4 rows), each nozzle actingas a separate pump and each row feeding from a separate reservoir, whichallows mixing of at least 4 different liquid streams as they aredeposited resulting in creation of one or multiple gradients duringdeposition. A multi-channel wash and refill position may be incorporatedinto the apparatus so that individual rows of nozzles can be washed andrefilled any number of times with new media as a part of the printingoperation. Current piezo-electric pumps can deposit 6 pL (6×10⁻¹² L) in45 micron droplets. The corresponding density of individual immobilizedcell drops one drop diameter apart is 12.3×10³ drops/cm² or 7.4×10⁶drops on an 8.5×11 inch sheet.

Using such printing techniques, a wide variety of reaction zones can becreated, each with its own micro-environment. For example, regions ofvarious antibiotic concentrations can be generated by depositingdifferent amounts of liquid from nozzles connected to separatereservoirs on a print head. Alternatively, a large number of plantpathogens can be printed and immobilized in a polymer matrix in anarray. Thus, a single plant leaf can be exposed to a large number ofplant pathogens simultaneously.

The devices may optionally include a removable film (a “top” film) thatprotects the biomaterial-containing layers. This top or protective filmis typically a layer of foil, although it could be a layer of celluloseacetate, or a wide variety of other synthetic or natural materials. Thedevices may also optionally include a removable film (a “bottom” orprotective film) that protects the supporting substrate, such as alight-sensitive electronic chip.

Various embodiments of the devices of the present invention aredescribed by reference to the figures. Many of these devices aredescribed with respect to the detection of mercury (whether it be in theform of inorganic mercury such as Hg²⁺ or organic mercury such asmonomethyl mercury), although other metals or organic materials could bedetected. Also, many of these devices are described with respect to thedetection of mercury in fish tissue or fluids, although other samplescould be tested. Furthermore, many of the embodiments include cells thatemit light upon exposure to mercury. Again, this is only forillustration purposes as other types of cells or other biologicalmaterials can be used and the devices modified accordingly, which wouldbe readily apparent to one of skill in the art upon reading theteachings herein.

FIG. 1A shows a basic structure for a device according to the presentinvention. The device 10 includes a biostructure 12, which is in theform of a biomaterial-containing layer. This includes immobilized cellsand is also referred to herein as a cell-containing indicator coating.The biostructure 12 is disposed on a photosensitive film 14 (thesupporting substrate). The device also includes removable protectivefilms 16 (first or top film) and 18 (second or bottom film), which maybe made of foil and may or may not include pull tabs (which is shown forfilm 16). FIG. 1B is a bottom view of the photosensitive film 14 (suchas a commercially available sheet of POLAROID film) after the film 18 isremoved showing a photosensitive area 17, which displays a response tomercury. Also shown is a built-in photodensity measuring device 19,which is used as a comparator. For example, the photodensity measuringdevice 19 can be a strip of material, such as paper or plastic, havingprinted thereon an image with increasing black density graduated tocorrespond to the level of mercury detected. The photodensity measuringdevice 19 also could be a mercury-containing compound spread on thesurface at an increasing concentration. In this way, the device wouldinclude an internal standard for quantifying the level of mercury. Thus,in both cases the strip would be labeled with the mercury concentration.In use, the top film 16 is removed to expose the immobilized cells tothe sample of interest. The immobilized cells, sample of interest, andnutrients (such as glucose in the presence of a buffer, for example) areincubated (for example, under ambient conditions for at least about 15minutes). The bottom film 18 is then removed and the level of mercury(which may be in the form of inorganic mercury and/or monomethylmercury) is determined by viewing the photosensitive area 17 andcomparing it to the photodensity measuring device 19.

FIG. 2A shows an alternative construction for a device 20 in which abiostructure 22 (in the form of a biomaterial-containing layer havingimmobilized cells therein) is disposed on a light-sensitive electronicchip 24. The device also includes removable protective film 26, whichmay or may not include a pull tab (which is shown for film 26). FIG. 2Bis a bottom view of the electronic chip 24, which may be reusable, andhas an electrical connection 25. In use, the film 26 is removed toexpose the immobilized cells to the sample of interest. The immobilizedcells, sample of interest, and nutrients are incubated as describedabove. The level of mercury is measured with a voltmeter (not shown).The light generated by the cells is measured by the voltage generated bythe light sensitive chip.

FIG. 3 shows an alternative structure for a device 30 in which abiostructure 32 (in the form of a biomaterial-containing lazer havingimmobilized cells therein) is disposed on a substrate 34 that does notdetect the signal (e.g., light, heat, etc.) produced by the cells. Thesubstrate shown is not light transmissive, although this is not anecessary requirement. The device also includes removable protectivefilm 36, which may or may not include a pull tab (which is shown forfilm 36). In use, the film 36 is removed to expose the immobilized cellsto the sample of interest. The immobilized cells, sample of interest,and nutrients are incubated as described above. The light generated bythe cells is measured with an external meter, such as a scintillationcounter or light meter. The mercury is correlated to the light level.Hand-held or laboratory meters are available.

FIG. 4 shows a device 40 that can penetrate into a sample of interest,such as fish tissue, through the scales, skin, and into the tissue. Thepenetration device 40 includes the construction shown in FIG. 1, whichincludes a biostructure 42 disposed on a photosensitive film 44 (oralternatively, it could include the constructions shown in FIG. 2 or 3).The device also includes removable bottom film 48 (and a top film, whichis not shown). In use, a cut is made in the fish, the top film (notshown) that protects the biostructure 42 is removed, device 40 isinserted into the cut and placed there for a predetermined length oftime to sorb the mercury. The device is removed and incubated withnutrients as described above. The level of mercury is then measured byevaluating the photosensitive film 44.

FIG. 5 shows yet another construction for a device 50 according to thepresent invention. The device 50 is in the form of a container, such asa pouch. It includes a biostructure 52 (having immobilized cellstherein) disposed on a supporting substrate 54, such as a photosensitivefilm or a light-sensitive electronic chip, for example. The device alsoincludes removable protective films 56 and 58. The top film 56 is not indirect contact with the biostructure 52; rather, it is spaced above it,thereby forming a cavity 59 for placement of a sample 57, such as aslice of fish tissue. In use, the film 56 is removed and the sample 57is placed in the cavity 59 in direct contact with the biostructure 52.The immobilized cells, sample, and nutrients are incubated as describedabove. The film 58 is then removed and the level of mercury is measuredusing the substrate 54.

FIG. 6 shows a very similar device to that shown in FIG. 5. In thisembodiment, the sample of interest, such as fish tissue need not beplaced in the cavity; rather an absorbent pad 65, which can be made ofcellulose or sponge, for example, containing fluids from the fish tissuecan be used. The device 60 includes a biostructure 62 disposed onsupport 64, which can be a photosensitive film or a light-sensitiveelectronic chip, for example. The device also includes removableprotective films 66 and 68. As in the device shown in FIG. 5, the topfilm 66 is not in direct contact with the biostructure 62; rather, it isspaced above it, thereby forming a cavity 69 for placement of anabsorbent pad 65. In use, the film 66 is removed and the absorbent pad65, which had been in contact with a slice of fish 67 to absorb fluidsfrom the fish tissue, is placed in the cavity 69 in direct contact withthe biostructure 62. The immobilized cells, absorbent pad, and nutrientsare incubated as described above. The film 68 is then removed and thelevel of mercury is measured using the support 64.

FIG. 7A shows a device 70 that includes the construction shown in FIG. 1(or alternatively, it could include the constructions shown in FIG. 2 or3), that includes a coring device 71 that is inserted into a container73. The container 73 includes a reservoir 75, which is filled withnutrients and buffer. The bottom of the container 73 (or optionally thesides of the container) includes a biostructure 72 disposed on aphotosensitive film 74. As shown in FIG. 7B, the coring device 71 isdesigned to penetrate fish scales, skin, and tissue, as described abovefor FIG. 4. It is used to remove a core sample of fish tissue anddeliver this core sample to the reservoir 75 in container 73. Both thecoring device 71 and the container 73 can have threads such that thecoring device 71 is screwed into the container, although this is not arequirement. The mercury from the tissue sample migrates to theimmobilized mercury sensitive cells located in the biostructure 72 inthe bottom (or sides) of the container 73. The reservoir 75 includesnutrients in a buffer for incubation of the cells. The light generatedby the cells is detected by a photosensitive film 74 (e.g., POLAROIDfilm) attached to the bottom (or sides) of the nutrient reservoir 75.

FIG. 8A shows a device similar to that shown in FIG. 7, except that thecoring device 81 includes a biostructure 82 (having immobilized cellstherein) directly on the coring device 81. Also, the coring device 81does not need to be hollow. Thus, it does not necessarily remove a coretissue sample from a fish as described with respect to FIG. 7. In thisembodiment and as shown in FIG. 8B, the immobilized cells located in thetip of the coring device 81 are exposed to the fish tissue while it isinserted in the fish tissue. After exposure to the fish tissue, thecoring device 81 is inserted into the reservoir-85 of container 83 wherethe cells are incubated in buffer and nutrients. The light generated bythe cells is detected by a photosensitive film 84 (e.g., POLAROID film)attached to the bottom (could also be the sides) of the nutrientreservoir 85.

FIG. 9 shows a device that allows for a sample to be drawn into areservoir, such as into a syringe. In device 90 there is a constructionas described above for FIG. 1. In FIG. 9A, a side view shows across-section of the device 90 that includes a biostructure 92 coated ona photosensitive film 94. The device also includes removable protectivefilm 98. The device 90 also includes a syringe needle 91, a reservoir93, and a bulb 95, which can be used to draw up a liquid sample into thereservoir 93. The biostructure 92 is positioned inside the reservoirwith photosensitive film 94 and protective film 98 forming an externalwall. FIG. 9B is a bottom view of the photosensitive film 94 (e.g.,POLAROID film) after the film 98 is removed showing a photosensitivearea 97, which displays a response to mercury. Also shown is a built-inphotodensity measuring device 99 as described above with respect toFIG. 1. In use, a liquid sample is drawn into the reservoir 93, where itcomes in contact with the immobilized cells of the biostructure 92.Nutrients are added and the cells and sample are incubated. The film 98is then removed and the level of mercury is measured by evaluating thephotosensitive area 97 as it compares to the photodensity measuringdevice 99.

FIG. 10 discloses a device that includes a biostructure in the form of acell-containing indicator coating coated on a fiber 100, composed of,for example, polyester, nylon, cellulose acetate, or an optical fiber.This fiber can be an optical fiber, for example. As shown in FIG. 10A,the coated fiber 100 has an end-stop 101 and a penetrating end 103. Thedevice is shown penetrating through a fish tail. Once a sufficientamount of time has lapsed for the immobilized cells of thecell-containing indicator to be in contact with the fish tissue, thecoated fiber 100 is removed. As shown in FIG. 10B, the coated fiber 100is then placed in contact with a photosensitive film 104, which can bein a pouch or container, for example, having nutrients and buffertherein. After a sufficient incubation period, the mercury can bequalitatively detected by viewing a photographic image 105 of the threadon the photosensitive film 104.

FIG. 11A shows a cross-section of a coated fiber 100, as shown in FIG.10. The coated fiber 100 includes a monofilament 106 coated with abiostructure 102. The biostructure 102 includes a cell-containingpolymeric layer 107 and a polymeric overlayer 108. FIG. 11B shows across-section of a coated fiber 110 that includes a multiple filamentthread 116 with a biostructure 112. The biostructure 112 includes acell-containing polymeric layer 117 and a polymeric overlayer 118. Thecoated fiber 110 may also include a polymeric precoat layer 119 betweenthe multiple filament thread 116 and the cell-containing polymeric layer107.

FIG. 12 shows a device 120 in which cells are used that contain aplasmid with a mercury resistance promoter that activates a gene toproduce a secreted protease or lipase. The cells are included in abiostructure 122. A pop-up indicator rod 121 is initially held in placeby a protein or lipid based glue 123. The pop-up indicator rod 121 maybe used either qualitatively or quantitatively, if it includes anindicator scale (not shown). The device also includes a spring 125 undertension that is attached to the pop-up indicator rod 121. In use, thedevice 120 is inserted into fish tissue. When mercury is detected by thecells in the biostructure 122 as it diffuses through a perforatedhousing 126 of device 120, the resulting protease or lipase degrades theglue 122 and releases the rod 121. As a result of the tension placed onthe rod 121 by the spring 125, the rod moves up. By the design of therod, the glue, and the glue holder the device could be made to bequantitative. Alternately, the pop-up indicator rod 121 could beattached to a piston to measure the production of a gas. In that casethe cells would contain the genes needed to produce high levels of gaswhen activated. In an alternative embodiment, the biostructure couldinclude a protein or lipid based glue in place of, or in addition to,the polymer used to make the biostructure.

FIG. 13 is a schematic of a preferred template assembly used to makepatches of cell-containing indicator coatings with a top-coating. FIG.13A is a top-view and side-view of template assembly 130. The templateassembly 130 includes a template 131, which can be made of a variety ofmaterials, such as paper, and includes holes 132 punched therein. Thetemplate 131 (e.g., 42.4 microns thick) is disposed on a supportingsubstrate 133 (e.g., 48 microns thick). FIG. 13B is a top view and sideview of cell-coat patches 134 (e.g., 30 microns thick) with surroundingspacer 135 (e.g., 155 microns thick) and a gap 136 (to be filled by atop coat) therebetween. FIG. 13C is a top view and side view of finishedtop-coated patches that include cell-coated patches 134 on a substrate133 and a top or sealant coating 137 disposed on the cell-coated patches134 and substrate 133 in the gaps 136.

Another embodiment of the present invention is shown in FIG. 14. In thisembodiment, a polyester substrate 140 is coated with a biostructure 141.The biostructure 141 includes a porous latex layer 142 containing viableE. coli cells, a porous latex sealant coating 143, and a discontinuousnonporous latex layer 144 which form walls. FIG. 15 includes theselayers plus a porous latex layer 145 with a mercury absorbant materialdisposed between the porous sealant coating 143 and porous latex layer142 containing a viable E. coli cells.

Yet another embodiment of the present invention is shown in FIG. 16.This includes a polyester substrate 160 on which is disposed abiostructure 161. The biostructure 161 includes a discontinuous porouslatex layer 162 containing viable E. coli cells, a discontinuous porouslatex sealant coating 163, and a discontinuous nonporous latex layer164. These layers form a porous channel 166 between two wells 168. Theoutline of the nonporous latex template that forms the wells 168 andchannel 166 is represented by 167.

FIG. 17 represents a layered porous array of microwells withbidirectional channels for delivery of drug screening candidates. Thedevice is shown with four reaction zones. As shown in FIG. 17A (across-section taken along line A) the device includes impermeable topand bottom layers 179, y-direction feed channels 180, nonporous fillerlayers 181, waste channel 182 shown in the y-direction, but could be inthe x-direction also. A cross-section of the device taken along line B,shows a reaction zone of immobilized biological particles 183, and ax-direction feed channel 184. A cross-section of the device taken alongline C again shows the x-direction feed channels 184.

Devices containing 3-dimensional microstructures as shown in FIG. 17 canbe used for screening new drug candidates. For example, for cells thatproduce a candidate that needs to be screened against a large number oftargets, a microstructure could be designed in which a matrix ofcandidate-producing cells are immobilized on top of a polymer layercontaining a matrix of target molecules.

Objects and advantages of this invention are further illustrated by thefollowing examples, but the particular materials and amounts thereofrecited in these examples, as well as other conditions and details,should not be construed to unduly limit this invention.

EXAMPLES

Method for Preparing Mercury Biosensor Films of Escherichia coli

Bacterial Strains and Media. The methods procedures and techniques aresubstantially those found in well known molecular cloning and geneticsguides such as Maniatis et al., Molecular Cloning: a Laboratory Manual,Cold Spring Habor, N.Y., 1982. Materials are commercially available fromsources such as GIBCO/BRL, Gathersburg, Md., or Promega, Madison, Wis.E. coli HB101 containing pRB28, pOS14, or pOS15 are in the public butcould be constructed through these cloning methods as described bySelifonova et al., Appl. Environ. Microbiol., 59, 3083–3090, (1993)).

Coating Materials. Harvested Escherichia coli HB101 cells were mixedwith glycerol and acrylic/vinyl acetate copolymer latex (Rohm and Haas,Philadelphia, Pa.) in a ratio depending on the number of cellsimmobilized. Commonly used was 1.2 g cell paste, 0.3 ml 50% (w/w)glycerol, 1 ml latex, which were mixed together. The cell-polymermixture was coated onto a polyester template (e.g., pressure sensitiveadhesive tape) using a 26 mil wire wound rod (Mayer bar) at 4° C., asdescribed in greater detail below. After the coated layer was dry, thetemplate was removed, and a second layer of latex (overlayer) was coatedon top. The topcoat layer was dried at 4° C., and the assembly was curedat 37° C. for 30 minutes or shorter. Individual patches were excised andrehydrated in buffer (5 mM pyruvate, NaK-phosphate buffer pH 6.8 [34 mMsodium phosphate; 33 mM potassium phosphate, pH adjusted to 6.8] and0.091 mM (NH₄)₂SO₄). The preparation of the template and coatingprocedure is described in detail below and with reference to FIG. 13.

Coating Method. A punch was used to create circular holes in a pressuresensitive tape such as that obtained from Minnesota Mining &Manufacturing Company, St. Paul, Minn., under the trade designation SM7830 to form a template. The template was rolled onto a substrate ofsimilar length and width. This creates uniform circular wells of adesired diameter and depth (12.7 mm diameter and 42.6 μm well depth wascommonly used), as shown in FIG. 13A. A pool of cell-polymer mix wasdelivered at the top of the template in a line spanning the width of thepatch area. A 26 mil wire diameter Mayer rod was placed above and thendrawn through the coating liquid and across the wells formed by thetemplate. The cell-coat was dried until no moisture was visible. At 4°C. and 70% relative humidity, drying took 2 hours. The template was thenpeeled off. As shown in FIG. 13B, a spacer consisting of a pressuresensitive tape was placed around the newly formed cell-coat patches toprevent the Mayer rod from touching during coating. Commonly used spacerthickness was 155 μm. The spacer was laid down on all four sides of thesubstrate containing the cell patches to contain the top-coating liquidduring drying. A pool of sealant liquid (latex with 5% glycerol) wasdelivered in line spanning the width of the area enclosed by the spacerand coated with a 26 mil wire diameter Mayer rod. After the top-coat haddried, the spacer was removed, leaving the construction shown in FIG.13C. The top-coat was dried at 4° C. and 70% relative humidity for 2.5hours until it took on a transparent and matte appearance.

Detection of Mercury,

FIG. 18

Section A. Patches of immobilized E. coli HB101 harboring the pRB28mer-lux constructs were analyzed individually in 20 ml scintillationvials in a liquid scintillation counter, (single photon counting mode, 1minute counting time, Beckman, LS 7000, Columbia, Md.) for luciferaseactivity after exposure (the patches were submerged in the liquidcontaining the mercury) to HgCl₂ concentrations from 0.1 nM to 10,000nM. Luciferase activity induced by 0.1, 1, or 10 nM HgCl₂ was notapparent during the first 5 hours of induction but increasedsubstantially during the next 15 hours after which time the activitycontinued to increase or remained constant until 37 hours. E. coli HB101(pRB28) exposed to higher levels of Hg(II) had significantly differentkinetics of luciferase induction. At 100, 1,000 or 10,000 nM HgCl₂, theluciferase activity reached maximum detection levels (limited by thescintillation counter to 6×10⁶ count of single photons per minute)within the first 5 hours of induction.

Section B. Patches of immobilized E. coli HB101 harboring the pOS14mer-lux constructs were analyzed individually for luciferase activityafter exposure to HgCl₂ concentrations from 0.1 nM to 10,000 nM, at 0.1nM HgCl₂. Light induction was not significant compared to the control.Hg(II) concentrations at 1 nM and 10 nM induced luciferase activityafter a 4–5 hour lag, and luciferase activity increased during the next10 hours of the assay. At 100, 1,000 and 10,000 nM HgCl₂, luciferaseactivity was evident after 2 hours of incubation and reached the maximumdetectable (6×10⁶ count of single photons per minute) after 5–8 hours.

Section C. Patches of immobilized E. coli HB101 harboring the pOS15mer-lux constructs were analyzed individually for luciferase activityafter exposure to HgCl₂ concentrations from 0.1 nM to 1,000 nM. The onlyconcentration of Hg(II) that showed significant luciferase inductionabove background levels with pOS15 was 1,000 nM HgCl₂.

FIG. 19

Patches containing E. coli HB 1101 (pRB28) cells that were identical tothose used for HgCl₂ induction of luciferase activity in pyruvate bufferwere stored at −20° C. for 3 months or as dry (meaning nonrehydrated)for 14 days. Samples were then either thawed or rehydrated in pyruvatephosphate buffer mentioned above and exposed to HgCl₂. The inducedmaximum luciferase activity was compared to freshly prepared immobilizedcell patches. Freezing the patches did not significantly affectluciferase induction. Maximum sensitivity for frozen stored patches was0.1 nM HgCl₂ equal to that of non-stored patches. Also the range (0.1 to100 nM HgCl₂) in which a detectable change in signal was observed wasunchanged. Storage 14 days dry decreased the maximum luciferase activityobserved between 100 nM and 0.1 nM HgCl₂. The maximum sensitivitydecreased from 0.1 nM to 1 nM HgCl₂. The range in which there was adetectable change in signal was expanded to the range 1 nM to 10,000 nMHgCl₂.

Method for Preparing 3D Microstructures

Bacterial Strains, Chemicals, Media, and Growth Conditions. Bacterialstrain: E. coli HB101 containing pRB28. Growth medium: Luria-Bertani(LB) medium (10 g/l tryptone (Difco), 5 g/l yeast extract (Difco), 5 g/lNaCl [analytical grade], pH 7.2) containing 50 μg/ml kanamycin (SigmaChemical Company, St. Louis, Mo.) at 30° C. E. coli was grown overnightin 300 ml growth medium in 2 liter Erlenmeyer flask at approximately 150rpm in a Labline shaker (model 3525CC) and were harvested bycentrifugation for 15 minutes at 2800×g. The cell pellet was washed inpyruvate buffer (5 mM pyruvate, NaK-phosphate buffer pH 6.8 [34 mMsodium phosphate; 33 mM potassium phosphate, pH 6.8] and 0.091 mM(NH₄)₂SO₄) before repelleting at 2800×g for 15 min.

Latex Materials. Porous sealant coating: Rovace SF091 containing 0.4volume sucrose per volume dry latex polymer. Nonporous coating material:Rovace SF091 with no additives. Porous channel material Rhopaque HP 1055containing 1.5 ml/ml JP1225 latex. Absorbant layer material (absorbentfor Hg²⁺ ions) Rovace SF091 containing 0.4 volume sucrose per volume drylatex polymer and ammonium pyrrolidine dithiocarbamate (APDC) 0.05 g/L.Cell coat material: 1.2 g of washed wet cell pellet gently resuspendedin 0.3 ml 50% (v/v) glycerol in water with 1 mL Rovace SF091 addedimmediately prior to coating.

Template, Mask, Spacer and Coating Materials. Coatings were cast onclear 2 mil polyester (3M, St. Paul, Minn.). Templates and masks weremade from pressure sensitive clear vinyl (42.6 μm) (Con-Tact, Stamford,Conn.) and spacers from Marker tape (155 μm) (TimeMed, Chatsworth,Calif.).

Preparing Templates and Masks. A template is a pressure sensitive tapewith sections cut out where the coating liquid is to contact theunderlying coating or substrate. A template can be applied on top of aclean substrate or on an already coated substrate. Masks are singlepieces of pressure sensitive tape placed on top of the substrate or oncoatings. After application of one or more layers on top of a templateor mask each template or mask can be removed to expose the layer(s)beneath. Each template or mask was generated by manually punching with a½ inch diameter punch (O'Brien Consolidated Industries, Lewiston, Me.)or cutting with a razor blade. Templates were generated by taping 5pieces of pressure sensitive tape (clear vinyl) to the backside of atemplate figure and each section was cut through the 5 template piecessimultaneously on a poly-vinyl chloride board. The individual templatesheets were separated and cleaned with KIMWIPEs to remove dust.Templates with nicks or tears were discarded since these would preventthem from separating from the substrate without tearing. Templates wereapplied onto the substrate or coating by rolling them onto it with ahard rubber roller (Orcon Corporation, Union City, Calif.). This methodcreated uniform sections for patches or channels with a depth of 42.6μm.

Coating of Latex Polymers. All coating layers were coated using wirewound rods (Mayer bars) with a wire diameter of 26 mil (R. D.Specialities, Webster, N.Y.). Coatings were created on a constantpressure draw down coating apparatus (R. D. Specialities, Webster, N.Y.)with a 31 cm×46 cm vacuum table (Paul N. Gardner Company, Inc, PomponaBeach Fla.) on top and dried at 18° C. for 30 minutes.

Detection of Luciferase Activity in Immobilized E. coli HB101. Patcheswith latex immobilized E. coli HB101 cells were exposed to HgCl₂ bysoaking them in 10 ml of pyruvate buffer (induction buffer) containing100 nM or 0 nM HgCl₂ in sterile glass scintillation vials. Hg(II)concentrations were confirmed by cold vapour atomic fluorescencespectroscopy CVAFS (Brooks Rand Model III, Seattle, Wash.). Immobilizedcell samples were incubated in triplicate at ambient temperature, andluciferase activity was detected as counts per minute of ATP-dependentphoton emission in a liquid scintillation counter (Beckman, LS 7000,Columbia, Md.).

Latex Biosensor Patch Shown in FIG. 14. This device was created bycoating a cell-latex mixture onto an 8 hole ½ inch diameter template ona polyester substrate. The coating was then dried. A sealant coating wascoated on top of the cell coating with the template still in place.Following drying of the sealant coating the template was removed leavingtwo layered patches of approximately 60 micron thickness on thesubstrate. A mask consisting of ½ inch circles were applied to eachpatch. A spacer was laid around the patches on each side to preventcontact between the Mayer rod and the masks during coating. A nonporouscoating was subsequently coated on top of the masked patches and driedbefore removal of the masks. Induction at 100 nM Hg²⁺ resulted in aphoton emission count of 500,000 counts per minute resulting from themercury induced expression of luciferase. Induction at 0 nM Hg²⁺resulted in less than 61 photon counts per minute.

Latex Biosensor Patch Shown in FIG. 15. This device was created bycoating a cell-latex mixture onto an 8 hole ½ inch diameter template ona polyester substrate. The coating was then dried. An absorbent coatingwas coated on top and dried. A sealant coating was coated on top of theabsorbent coating with the template still in place. Following drying ofthe sealant coating the template was removed leaving three layeredpatches of approximately 90 micron thickness on the substrate. A maskconsisting of ½ inch circles were applied to each patch. Surrounding thepatches on each side a spacer was laid down to prevent contact betweenthe Mayer rod and the masks during coating. A nonporous coating wassubsequently coated on top of the masked patches and dried beforeremoval of the masks. Induction at 100 nM Hg²⁺ resulted in a photonemission count of less than 1000 counts per minute resulting from themercury induced expression of luciferase. The result demonstrated thatthe absorbent layer reduced the induced activity by 500 times. Inductionat 0 nM Hg²⁺ resulted in less than 50 photon counts per minute.

Latex Biosensor Patch Shown in FIG. 16. This device was created bycoating a cell-latex mixture onto an 8 hole ½ inch diameter template ona polyester substrate. The coating was then dried. A sealant coating wascoated on top of the cell coating with the template still in place.Following drying of the sealant coating the template was removed leavingtwo layered patches of approximately 60 micron thickness on thesubstrate. A second template consisting of ½ inch by 1 inch rectangularholes was applied on top of the patches so that ¼ inch of the patcheswere covered and so that the open area connected to opposing patches. Aporous channel layer was coated on top of the second template and dried.The second template was subsequently removed. A mask consisting of ½inch circles was applied to each patch. A spacer was laid around thepatches on each side to prevent contact between the Mayer rod and themasks during coating. A nonporous coating was subsequently coated on topof the masked patches and the porous channel layer and dried beforeremoval of the masks. Each patch with its channel was subsequentlyexcised so that each patch had a ½ inch channel attached. 5 mm of thechannel end was placed in induction buffer, leaving the circular partwith cells out of direct contact with the induction buffer. Induction at100 nM Hg²⁺ resulted in a photon emission count of more than 50,000counts per minute resulting from the mercury induced expression ofluciferase. Induction at 0 nM Hg²⁺ resulted in less than 110 photoncounts per minute.

Latex Biosensor Patch as in FIG. 14 Created on a Piezo-Electric Ink-JetPrinter. This sample was created by coating a cell-latex mixture andnonporous latex concurrently using an ink-jet printer (Canon PJ-1080a)with a piezo-electric print head. The cell latex mixture was loaded intothe yellow color ink reservoir, the nonporous latex was loaded into theblack color ink reservoir, and the porous latex mixture was loaded intothe blue color ink reservoir. Patches were coated by printing 4 times ontop of the same area a cell-latex center surrounded by a nonporous latexlayer. Each coating step was followed by a drying step prior to coatingagain. Total patch thickness was approximately 3 microns after 4coatings. Following the last printing and drying step a nonporoussealant coating was printed on top of the entire coated area. Fourdifferent patch sizes were made. Patch sizes were 9 mm×9 mm, 4.5 mm×4.5mm, 1.28 mm×0.96 mm, and 0.64 mm×0.64 mm. Induction at 100 nM Hg²⁺resulted in a photon emission count above noninduced patches of the samesize of 99,980 cpm for 9×9 mm patches, 767 cpm for 4.5×4.5 mm patches,11 cpm for 1.28×0.96 mm patches, and 3 cpm for 0.64×0.64 mm patches.

The complete disclosures of the patents, patent documents, andpublications cited herein are incorporated by reference in theirentirety as if each were individually incorporated. Variousmodifications and alterations to this invention will become apparent tothose skilled in the art without departing from the scope and spirit ofthis invention. It should be understood that this invention is notintended to be unduly limited by the illustrative embodiments andexamples set forth herein and that such examples and embodiments arepresented by way of example only with the scope of the inventionintended to be limited only by the claims set forth herein as follows.

1. A composite biological device comprising a biostructure comprising atleast one biological material as an integral imbedded component withinthe biostructure, wherein at least a portion of the biostructurecomprises a nonporous latex-derived material and at least a portion ofthe biostructure comprises a porous latex-derived material having the atleast one biological material imbedded therein, wherein the biologicalmaterial is a cell or microbe and metabolically active or becomesmetabolically active upon hydration, and further wherein thebiostructure is obtainable by gravure coating, piezo-electric printing,or acoustic printing.
 2. The composite device of claim 1 wherein thebiological material comprises a prokaryote, a eukaryote, an archeanorganism, or a combination thereof.
 3. The composite device of claim 1wherein the biological material comprises a mammalian cell, a bloodcell, an avian cell, a plant cell, an insect cell, a bacteriophage, aspore, a virus, or a combination thereof.
 4. The composite device ofclaim 1 wherein the biological material comprises a recombinantbacterial, yeast, or fungal cell.
 5. The composite device of claim 4wherein the recombinant cell is desiccation tolerant.
 6. The compositedevice of claim 1 wherein the biostructure further comprises at leastone additive selected from the group of a salt, a pigment, an adsorbent,a liquid crystal, a porosity modifier, a chelating agent, a nutrient, asurfactant, a dye, a photoreactive compound, an antibiotic, anantimicrobial, a bacteriostatic compound, an enzyme, an osmoprotectant,a biopolymer, a metal, a chemical catalyst, and a combination thereof.7. The composite device of claim 1 wherein the biostructure furthercomprises a transmitter incorporated therein.
 8. A method of determiningthe presence of an analyte in a sample, the method comprises contactingthe sample with the device of claim 7 wherein, upon contact with theanalyte, the biological material produces a response and emits a signal;and detecting the signal.
 9. The composite device of claim 1 wherein thebiostructure further comprises a detector incorporated therein.
 10. Thecomposite device of claim 9 wherein the detector senses a responseemitted from the biological material when in contact with an analyte.11. A method of determining the presence of an analyte in a sample, themethod comprises contacting the sample with the device of claim 9wherein, upon contact with the analyte, the biological material producesa response and emits a signal; and detecting the signal.
 12. Thecomposite device of claim 1 wherein the latex-derived material iscross-linked.
 13. A method of determining the presence of an analyte ina sample, the method comprises contacting the sample with the device ofclaim 12 wherein, upon contact with the analyte, the biological materialproduces a response and emits a signal; and detecting the signal. 14.The composite device of claim 1 wherein the biostructure is non-hydratedand the biological material becomes metabolically active upon hydration.15. The composite device of claim 1 wherein the porous latex-derivedmaterial comprises a mixture of latices.
 16. The composite device ofclaim 1 wherein the biostructure is supported on a substrate distinctfrom the nonporous latex-derived material.
 17. The composite device ofclaim 16, wherein the substrate comprises a membrane, a filament, awire, a film, a foam, a metal, a glass, a ceramic, an organic polymer,an electrode, a semiconductor device, or combinations thereof.
 18. Thecomposite device of claim 16 wherein the substrate comprises a metal ora polymeric material.
 19. The composite device of claim 16 wherein thesubstrate is an electronic device.
 20. The composite device of claim 1wherein the biostructure comprises wires or electrodes.
 21. A method ofdetermining the presence of an analyte in a sample, the method comprisescontacting the sample with the device of claim 20 wherein, upon contactwith the analyte, the biological material produces a response and emitsa signal; and detecting the signal.
 22. The composite device of claim 1wherein the biostructure is no greater than about 500 microns thick. 23.The composite device of claim 1 wherein the entire device is no greaterthan about 500 microns thick.
 24. A method of determining the presenceof an analyte in a sample, the method comprises contacting the samplewith the device of claim 1, wherein, upon contact with the analyte, thebiological material produces a response and emits a signal; anddetecting the signal.
 25. The composite device of claim 1 wherein thebiostructure comprises a porous sealant layer that does not includebiological material.
 26. A composite biological device comprising alayered biostructure comprising at least one biological material as anintegral component within the biostructure, wherein the biostructurecomprises: at least one layer comprising a porous latex-derived materialhaving the at least one biological material imbedded therein, and atleast one layer comprising a nonporous latex-derived material, whereinthe biological material is a cell or microbe and metabolically active orbecomes metabolically active upon hydration, and further wherein thebiostructure is obtainable by gravure coating, piezo-electric printing,or acoustic printing.
 27. A method of determining the presence of ananalyte in a sample, the method comprises contacting the sample with thedevice of claim 26 wherein, upon contact with the analyte, thebiological material produces a response and emits a signal; anddetecting the signal.
 28. A composite biological device comprising abiostructure comprising at least one biological material as an integralcomponent within the biostructure, wherein at least a portion of thebiostructure comprises a nonporous latex-derived material and at least aportion of the biostructure comprises a porous latex-derived materialhaving the at least one biological material imbedded therein, whereinthe biological material is a cell or microbe and metabolically active orbecomes metabolically active upon hydration, wherein the nonporousmaterial defines at least one channel or at least one well, and furtherwherein the biostructure is obtainable by gravure coating,piezo-electric printing, or acoustic printing.
 29. A method ofdetermining the presence of an analyte in a sample, the method comprisescontacting the sample with the device of claim 28, wherein, upon contactwith the analyte, the biological material produces a response and emitsa signal; and detecting the signal.
 30. A composite biological devicecomprising a biostructure comprising at least one biological material asan integral component within the biostructure, wherein at least aportion of the biostructure comprises a nonporous latex-derived materialand at least a portion of the biostructure comprises a porouslatex-derived material having the at least one biological materialimbedded therein, wherein the biological material is a cell or microbeand metabolically active or becomes metabolically active upon hydration,wherein the biostructure comprises no greater than about 75% by volumebiological material, and further wherein the biostructure is obtainableby gravure coating, piezo-electric printing, or acoustic printing. 31.The composite device of claim 30 wherein the biostructure comprises nogreater than about 50% by volume biological material.
 32. A method ofdetermining the presence of an analyte in a sample, the method comprisescontacting the sample with the device of claim 30, wherein, upon contactwith the analyte, the biological material produces a response and emitsa signal, and detecting the signal.
 33. A composite biological devicecomprising a three-dimensional biostructure comprising at least onebiological material as an integral, imbedded, permanently trapped,component within the biostructure, wherein at least a portion of thebiostructure comprises a nonporous latex-derived material and at least aportion of the biostructure comprises a porous latex-derived materialhaving the at least one biological material imbedded therein, whereinthe biological material is a cell or microbe and metabolically active orbecomes metabolically active upon hydration, and further wherein thebiostructure is obtainable by gravure coating, piezo-electric printing,or acoustic printing.
 34. A composite biological device comprising abiostructure comprising at least one biological material as an integralimbedded component within the biostructure, at least one porositymodifier, and at least one osmoprotectant, wherein at least a portion ofthe biostructure comprises a nonporous latex-derived material and atleast a portion of the biostructure comprises a porous latex-derivedmaterial having the at least one biological material imbedded therein,and further wherein the biological material is a cell or microbe andmetabolically active or becomes metabolically active upon hydration. 35.The composite device of claim 34 wherein the porosity modifier issucrose.
 36. The composite device of claim 34 wherein the osmoprotectantis glycerol.
 37. A method of determining the presence of an analyte in asample, the method comprises contacting the sample with the device ofclaim 34 wherein, upon contact with the analyte, the biological materialproduces a response and emits a signal; and detecting the signal.
 38. Acomposite biological device comprising a biostructure comprising atleast one biological material as an integral imbedded component withinthe biostructure, wherein at least a portion of the biostructurecomprises a nonporous latex-derived material and at least a portion ofthe biostructure comprises a porous latex-derived material having the atleast one biological material imbedded therein, further wherein thebiological material is a cell or microbe and metabolically active orbecomes metabolically active upon hydration, and further wherein thedevice is stable for at least 6 months under ambient conditions.
 39. Amethod of determining the presence of an analyte in a sample, the methodcomprises contacting the sample with the device of claim 38 wherein,upon contact with the analyte, the biological material produces aresponse and emits a signal; and detecting the signal.